Bleeding is a common clinical problem. It is a consequence of disease, trauma, surgery and medicinal treatment. It is imperative to mechanically stop the bleeding. This may be difficult or even impossible due to the location of the bleeding or because it diffuses from many (small) vessels. Patients who are bleeding may thus require treatment with agents that support haemostasis. This may be blood-derived products (haemotherapy), agents that cause the release of endogenous haemostatic agents, recombinant coagulation factors (F), or agents that delay the dissolution of blood clots.
The first line treatment among the blood derived products, often obtained from the local hospital, are whole blood for volume substitution and support of haemostasis, packed red cells for the improvement of oxygen transporting capacity, platelet concentrates to raise the number of platelets (if low or defective) and fresh frozen plasma for support of the haemostasis (blood coagulation and platelet aggregation). Second line plasma derived products that support haemostasis are plasma cryoprecipitate, prothrombin complex concentrates, activated prothrombin complex concentrates and purified coagulation factors. Several coagulation factors are today available as human recombinant proteins, inactive (coagulation factors VIII and IX) and activated (coagulation factor VIIa).
Haemophilia is an inherited or acquired bleeding disorder with either abnormal or deficient coagulation factor or antibodies directed towards a coagulation factor which inhibits the procoagulant function. The most common haemophilias are haemophilia A (lack coagulation factor VIII) and haemophilia B (factor IX). The purified or recombinant single coagulation factors are the main treatment of patients with haemophilia. Patients with inhibitory antibodies posses a treatment problem as they may also neutralise the coagulation factor that is administered to the patient.
The active form of Protein C (APC) is an inhibitor of plasma coagulation by degradation of the activated coagulation factors Va and VIIIa. Recombinant APC has been shown to be an effective treatment of undue plasma coagulation in patients with sepsis.
Coagulation factors for therapeutic use can be obtained from human plasma, although the purification process is not simple and requires many steps of which several aim at eliminating contaminating viruses. But even with extensive safety measures and testing of blood-derived products, contamination with infectious viruses or prions cannot be ruled out. Because of this risk it is highly desirable to produce human therapeutic proteins from recombinant cells grown in media without animal derived components. This is not always straightforward as many proteins require a mammalian host to be produced in a fully functional form, i.e. be correctly post-translationally modified. Among the coagulation factors commercially produced in recombinant cells are FVII (NovoSeven), FVIII (Kogenate, Recombinate, Refacto) and FIX (BeneFix) (Roddie and Ludlam. Blood Rev. 11:169-177, 1997) and Active Protein C (Xigris). One of the major obstacles in obtaining large amounts of fully functional recombinant human coagulation factors lies in the Gla-domain present in FII, FVII, FIX, FX, Protein S and Protein C. This domain contains glutamic acid residues that are post-translationally modified by addition of carboxyl groups. The production of these factors are hampered by the fact that over-expression of them result in under-carboxylated, and hence inactive, protein. The Gla modifications are a result of the action of a vitamin K-dependent enzyme called γ-glutamyl carboxylase (GGCX). This enzyme has been extensively studied by many scientists, particularly those involved in coagulation factor research (WO-A-8803926; Wu et al. Science 254(5038):1634-1636, 1991; Rehemtulla et al, Proc Natl Acad Sci USA 90:4611-4615, 1993; Stanley J. Biol. Chem. 274(24):16940-16944, 1999; Vo et al., FEBS letters 445:256-260, 1999; Begley et al, The Journal of Biological Chemistry 275(46):36245-36249, 2000; Walker et al., The Journal of Biological Chemistry 276(11):7769-7774, 2001; Bandyopadhyay, et al. Proc Natl Acad Sci USA 99(3):1264-1269, 2002; Czerwiec et al., Eur J Biochem 269:6162-6172, 2002; Hallgren et al, Biochemistry 41(50):15045-15055,2002; Harvey et al., The Journal of Biological Chemistry 278(10):8363-8369, 2003). Attempts to co-express GGCX with coagulation factor FIX has been tried by at least two scientific groups but were not successful (Rehemtulla, et al. 1993, ibid; Hallgren et al. 2002, ibid). Considering the large interest in GGCX enzymes, it may be assumed that many more trials have failed and thus have not been reported. GGCX requires reduced vitamin K as a cofactor. The reduced vitamin K is by GGCX converted to vitamin K epoxide, which is recycled to reduced vitamin K by Vitamin K epoxidoreductase (VKOR). Thus for efficient vitamin K dependent carboxylation of proteins two enzymes are required, GGCX and VKOR. Cloning and identification of VKOR was reported 2004 (Li et al, Nature 427:541-543, 2004, Rost et al., Nature 427:537-541, 2004). The VKOR protein is a 163 amino acid polypeptide with at least one predicted transmembrane region. From recombinant cells expressing VKOR activity is localized to the microsomal subcellular fraction.
For human FII (prothrombin) at least 8 out of 10 Glu residues have to be correctly modified in order to obtain fully functional prothrombin (Malhotra, et al., J. Biol. Chem. 260:279-287, 1985; Seegers and Walz Prothrombin and other vitamin K proteins', CRC Press, 1986). Similarity, human coagulation factor IX clotting activity require γ-carboxylation of at lest 10 out of 12 glutamic residues in the Gla-domain (White et al, Thromb. Haemost. 78:261-265, 1997). Extensive efforts to obtain high production levels of rhFII have been made using several different systems such as CHO cells, BHK cells, 293cells and vaccinia virus expression systems, but have all failed or resulted in an under-carboxylated product and thus functionally inactive prothrombin (Jørgensen et al., J. Biol. Chem. 262:6729-6734, 1987; Russo et al., Biotechnol Appl Biochem 14(2):222-233, 1991; Fischer et al, J Biotechnol 38(2):129-136, 1995; Herlitschka et al. Protein Expr. Purif. 8(3):358-364, 1996; Russo et al, Protein Expr. Purif. 10:214-225, 1997; Vo et al. 1999, ibid; Wu and Suttie Thromb Res 96(2):91-98, 1999). Earlier reported productivities for carboxylated recombinant human prothrombin are low; 20 mg/L for mutant prothrombin (Côte et al., J. Biol. Chem 269:11374-11380, 1994), 0.55 mg/L for human prothrombin expressed in CHO cells (fully carboxylated, Jøergensen et al. 1987, ibid), 25 mg/L in CHO cells (degree of carboxylation not shown, Russo et al. 1997, ibid).
As far as known co-expression of a protein requiring γ-carboxylation and VKOR has not been reported earlier.
WO 92/19636 discloses the cloning and sequence identification of a human and bovine vitamin K dependent carboxylase. The application suggests co-expressing the vitamin K dependent carboxylase and a vitamin K dependent protein in a suitable host cell in order to prepare the vitamin K dependent protein. No co-expression of the carboxylase and vitamin K dependent protein is exemplified.
WO 92/19636 discloses the cloning and sequence identification of a human and bovine vitamin K dependent carboxylase. The application suggests co-expressing the vitamin K dependent carboxylase (GGCX) and a vitamin K dependent protein in a suitable host cell in order to prepare the vitamin K dependent protein. No co-expression of the carboxylase and vitamin K dependent protein is exemplified.
WO 2005/038019 claims a method of increasing the overall productivity of γ-carboxylated protein by a controlled co-expression of γ-carboxylated protein and GGCX. The invention is exemplified with improved productivity of coagulation factors II and FIX.
WO 2005/030039 suggests co-expression of vitamin K dependent proteins with Vitamin K epoxide reductase (VKOR) in order to improve γ-carboxylation. However, no such co-expression expression is exemplified.
Co-expression of coagulation factor X (FX) and VKOR has been shown to improve the share of γ-carboxylated protein by Sun et al. (Blood 106: 3811-3815, 2005). Wajih et al. (JBC 280:31603-31607, 2005) has in addition demonstrated improved share of γ-carboxylated coagulation factor IX (FIX) by co-expression with VKOR. Both publications reported that VKOR incresed the share of γ-carboxylated protein but VKOR co-expression did not improve the overall productivity of coagulation factor.
There is a need for improved methods to produce activated blood clotting factors in high yields. The present invention sets out to address this need.